PT1864666E - Protective agent for retinal neuronal cell containing prostaglandin f2 alpha derivative as active ingredient - Google Patents

Description

DESCRIPTION OF THE PREFERRED EMBODIMENT OF RETINAL NEURONAL CELL CONTAINING PROSTAGLANDIN F2 ALPHA DERIVATIVE AS ACTIVE INGREDIENT " The present invention relates to a protective agent for a retinal neuronal cell containing a prostaglandin F 2 O derivative as an active ingredient for use in preventing or treating an eye disease associated with retinal neuronal cell damage. The retina is a tissue having a thickness of from 0.1 to 0.5 mm consisting of ten layers of internal limiting membrane, fibrous nervous layer, ganglionic cell layer, inner plexiform layer, inner nuclear layer, outer plexiform layer, outer nuclear layer, outer limiting membrane, photo-receptor cell layer and retinal pigment epithelium layer, and there are present retinal neuronal cell groups including photo-receptor cells, bipolar cells, ganglion cells, horizontal cells, akcrine cells and Muller cells.

Retinal neuronal cells play an important role in receiving and transmitting visual information, such as converting light stimulation into an electrical signal and transmitting the signal to the brain.

To specifically describe the mechanism of such transmission, visual information from the eyes is converted into an electrical signal through photo-receptor cells and transmitted to ganglion cells by means of horizontal cells, bipolar cells and / or a-lactic cells. Thereafter, the electrical signal is transmitted to the brain by means of the optic nerve which is a bundle of optic nerve fibers including ganglion cell axons.

On the other hand, when these retinal neuronal cells are damaged due to various causes, homeostasis (a function of oxygen delivery or nutrition to retinal neuronal cells through the retinal blood circulation and the like) of retinal neuronal cells can not be maintained and transmission of visual information to the brain is inhibited. For example, it is widely known that retinal neuronal cell dysfunction is elicited in various retinal diseases, such as retinal vascular occlusion, diabetic retinopathy, ischemic optic neuropathy, glaucoma, macular degeneration, retinitis pigmentosa and Leber's disease (Brain Res. Bull. , 62 (6), 447-453 (2004)).

Recently, retinal neuronal cell death due to retinal ischemia has been one of the causes of retinal neuronal cell injury and the following events have been reported in relation to retinal neuronal cell death due to retinal ischemia (JP-A-2003-146904 and Nature Rev., 2, 448-459 (2003)). 1) The mechanism of retinal neuronal cell death due to retinal ischemia is similar to that of cerebral neuronal cell death due to cerebral ischemia. 2 to layer 2) In short-term retinal ischemia, internal retinal (internal plexiform layer) is selectively damaged. 3) Excessive release of glutamate during retinal ischemia can be observed. 4) By injecting an excitatory amino acid, such as glutamate, into the vitreous body, retinal neuronal cell death is induced. 5) Retinal N-methyl-D-aspartate (NMDA) -mediated over-stimulation promotes calcium (Ca) influx into cells, which results in inducing cellular injury through induction of nitrogen monoxide (NO). From these facts, a drug, such as a glutamate neurotoxicity inhibitor and an NMDA receptor antagonist or an NO synthesis inhibitor, is considered useful for the treatment of an eye disease caused by retinal neuronal cell injury and have performed several studies.

For example, JP-A-2001-072591 discloses a protective agent for a retinal neuronal cell containing nipradilol which is one of the β-blockers as the active ingredient. WO 01/056606 discloses a protective agent for an optical ganglion cell containing an interleukin-1 receptor antagonist protein as the active ingredient. WO 03/004058 discloses a protective agent for an optic ganglion cell containing a ορ receptor antagonist, such as brimonidine hydrochloride as an active ingredient. Experimental Eye Res., 72, 479-486 (2001) discloses a nerve-protecting effect of latanoprost which is one of the prostaglandin derivatives, etc. 3

On the other hand, the prostaglandin F2a derivatives are disclosed as a therapeutic agent for glaucoma having an intraocular pressure reducing action in JP-A-59-1418, JP-T-3-501025, JP-T-8-501310, JP A-10-182465, WO 98/12175, European Patent Application Publication No. 850926, JP-A-2004-002462, JP-A-10-259179, JP-A-2002-293771 and JP-A- 2003-321442. JP-A-59-1418 discloses a natural prostaglandin F2a derivative. JP-T-3-501025 discloses a latanoprost-related compound. JP-T-8-501310 discloses a bimatoprost-related compound. JP-A-10-182465 discloses a travoprost-related compound. WO 98/12175 discloses a monofluoroprostaglandin derivative F2a. European Patent Application Publication No. 850926 and JP-A-2004-002462 disclose a difluoroprostaglandin derivative F2a. JP-A-10-259179 discloses a fluorine-containing F 2 O 2 prostaglandin derivative having a multisubstituted aryloxy group. JP-A-2002-293771 discloses an ether type difluoroprostaglandin derivative F2cx. JP-A-2003-321442 discloses an F2cx difluoroprostaglandin amide derivative.

However, the disclosure of these documents does not, at all, describe an effect of a fluorine-containing prostaglandin F2a derivative on the protection of a retinal neuronal cell. It is a very interesting subject to find a new pharmaceutical application of a prostaglandin F2a derivative (in particular, a fluorine-containing prostaglandin F2a).

Accordingly, the present applicants have conducted intensive studies in order to find a new pharmaceutical application 4 of a prostaglandin F2a derivative. As a result, they find that the prostaglandin F2cx derivative inhibits glutamate induced retinal neuronal cell death, in a concentration-dependent effect on rat fetal retinal neuronal cells, in other words, the prostaglandin F 2α derivative acts directly on retinal neuronal cells and exhibits a protective effect, thereby achieving the present invention. The present invention relates to a protective agent for a retinal neuronal cell containing a prostaglandin F 2 O derivative as an active ingredient for use in preventing or treating an eye disease associated with retinal neuronal cell damage.

In addition, the present invention may be used in a method of protecting a retinal neuronal cell and in a method of preventing or treating an ocular disease associated with retinal neuronal cell injury.

In the present invention, " Prostaglandin F2a derivative " means a prostaglandin F2a related compound derived from the prostanoic acid skeleton as defined in claim 1.

In general, a protective agent for a retinal neuronal cell is, for example, one containing a prostaglandin F 2 α derivative or a salt thereof, such as a natural prostaglandin F 2α derivative disclosed in JP-A-59-1418, a compound (with the proviso that a latanoprost-related compound or a salt thereof excludes latanoprost) disclosed in JP-T-3-501025, a compound related to bimatoprost (preferably bimatoprost or a salt thereof) disclosed in JP-T-8-501310, a compound related to travoprost (preferably travoprost or a salt thereof) disclosed in JP-A-10-182465 or a fluorine-containing prostaglandin F2a derivative disclosed in WO European Patent Application No. 850926, JP-A-2004-002462, JP-A-10-259179, JP-A-2002-293771 or JP-A-2003-321442 as the active ingredient.

Preferably, a protective agent for a retinal neuronal cell is, for example, one that contains a fluorine-containing prostaglandin F2a derivative. &Quot; as active ingredient. &Quot; Fluor containing prostaglandin F2a " means a prostaglandin F 2a derivative having one or more fluorine atoms.

Specifically, a protective agent for a retinal neuronal cell is, for example, one containing a fluorine-containing prostaglandin F2a derivative disclosed in WO 98/12175, European Patent Application Publication No. 850926, JP-A -2004-002462, JP-A-10-259179, JP-A-2002-293771 or JP-A-2003-321442 as the active ingredient.

More preferably, a protective agent for a retinal neuronal cell is, for example, one that contains a 15,15-difluoroprostaglandin derivative F2o1 disclosed in European Patent Application Publication No. 850926, JP-A-2004 -002462, JP-A-10-259179, JP-A-2002-293771 or JP-A-2003-321442 as the active ingredient.

According to the present invention, the protective agent for a retinal neuronal cell is one containing a 15,15-difluoroprostaglandin derivative F2o represented by the following general formula (1), which is an even more preferred fluorine-containing F2o prostaglandin derivative or a salt thereof, as an active ingredient.

HQ

(D

[R represents a carboxyl group or a salt thereof or an alkoxycarbonyl group.

The respective groups and terms defined in this description will be shown below. 0 " halogen " refers to fluorine, chlorine, bromine or iodine. 0 " alkyl " refers to straight or branched chain alkyl having 1 to 6 carbon atoms. Specific examples thereof include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, and the like. &Quot; alkoxy " refers to straight or branched chain alkoxy having 1 to 6 carbon atoms. Specific examples thereof include methoxy, ethoxy, n-propoxy, n-butoxy, n-pentyloxy, n-hexyloxy, isopropoxy, isobutoxy, sec-butoxy, tert-butoxy, isopentyloxy and the like. 0 " aryl " refers to monocyclic or bicyclic aromatic hydrocarbon or tricyclic condensed polycyclic aromatic hydrocarbon having 6 to 14 carbon atoms. Specific examples thereof include phenyl, naphthyl, anthryl, phenanthryl and the like. &Quot; aryloxy " refers to monocyclic, or bicyclic aromatic hydrocarbonoxyl or tricyclic condensed polycyclic aromatic hydrocarbon having 6 to 14 carbon atoms. Specific examples thereof include phenoxy, naphthyloxy, anthryloxy, phenanthryloxy and the like. 0 " alkylamino " refers to monoalkylamino or dialkylamino having 1 to 12 carbon atoms. Specific examples thereof include methylamino, ethylamino, dimethylamino, dihexylamino and the like. &Quot; arylamino " refers to monoarylamino or diarylamino having 6 to 28 carbon atoms. Specific examples thereof include phenylamino, naphthylamino, methylphenylamino, ethylphenylamino, diphenylamino, diantrilamino and the like.

A particularly preferred fluorine-containing prostaglandin F 2a derivative is, for example, a 15,15-difluoroprostaglandin derivative of the above-mentioned general formula (1) in which R represents a carboxyl group or a salt group thereof or an isopropoxycarbonyl group.

These prostaglandin F 2α derivatives may be in the form of a salt with an inorganic acid, such as hydrochloric acid, hydrobromic acid, iodide acid, nitric acid, sulfuric acid or phosphoric acid, an organic acid, such as acetic acid, fumaric acid, maleic acid, succinic acid or citric acid, an alkali metal such as lithium, sodium or potassium, an alkaline earth metal such as calcium or magnesium, ammonia or the like. These salts are also included in the present invention.

In the present invention, the " retinal neuronal cell " means a neuronal cell involved in the transmission of visual signal to the brain. Specifically, it means a photo-receptor cell, a horizontal cell, a bipolar cell, an optical ganglion cell, an amacrine cell or the like.

In the present invention, " ocular disease " means an ocular disease associated with retinal neuronal cell injury.

Specifically, it means an abnormal visual field, retinal vascular occlusion, diabetic retinopathy, ischemic optic neuropathy, glaucoma, macular degeneration, retinitis pigmentosa, Leber's disease or the like and, preferably, means an abnormal visual field, retinal vascular occlusion, diabetic retinopathy, ischemic optic neuropathy, macular degeneration, retinitis pigmentosa or Leber's disease. The protective agent for a retinal neuronal cell of the present invention may be administered orally or parenterally. Examples of the dosage form for administration include an ophthalmic droplet, an ophthalmic ointment, an injection, a tablet, a capsule, a granule, a powder and the like, and particularly preferably an ophthalmic droplet. Such preparation may be prepared by any of the widely used techniques, for example, a technique disclosed in JP-A-59-1418, JP-T-3-501025, JP-T-8-501310, JP-A-10- WO 98/12175, European Patent Application Publication No. 850926, JP-A-2004-002462, JP-A-10-259179, JP-A-2002-293771, JP-A-2003-321442, WO 02/22131 or the like.

For example, an ophthalmic drop can be prepared using a tonicity agent, such as sodium chloride or concentrated glycerin, a buffer, such as sodium phosphate or sodium acetate, a surfactant, such as polyoxyethylene and sorbitan monooleate, polyoxyl 40 or polyoxyethylene hydrogenated castor oil, a stabilizer, such as sodium citrate or sodium edetate, a preservative, such as benzalkonium chloride or paraben as required. The pH of the ophthalmic droplet is allowed provided it is within the range which is acceptable as an ophthalmic preparation. The preferred pH is in the range of 4 to 8.

An ophthalmic ointment can be prepared using a widely used base, such as soft white paraffin or liquid paraffin, as required.

In addition, an oral preparation, such as a tablet, a capsule, a granule, a powder, may be prepared using an additive, such as lactose, crystalline cellulose, starch or a vegetable oil, a lubricant, such as magnesium stearate or talc, a binder, such as hydroxypropylcellulose or polyvinylpyrrolidone, a disintegrant, such as low-substituted carboxymethylcellulose calcium or hydroxypropylmethylcellulose, a coating agent, such as hydroxypropylmethylcellulose, macrogol or a silicone resin, a film forming agent such as gelatin or the like, according to need. The dose may be selected, appropriately, depending on the symptoms, age, dosage form and the like. An ocular drop may be instilled one to several times a day, at a concentration of from 0.00001 to 1% (w / v), preferably from 0,0001 to 1% (w / v). An oral preparation may be administered once or divided several times in a dose, generally from 0.01 to 5000 mg per day, preferably from 0.1 to 1000 mg per day.

As will be described in detail in the Pharmacological Test section below, an effect of a prostaglandin F2a derivative on glutamate induced retinal neuronal cell death was examined using rat fetal retinal neuronal cells. As a result, the prostaglandin F2a derivative inhibited glutamate induced retinal neuronal cell death in a concentration-dependent effect. That is, the prostaglandin F2a derivative has a protective action of a retinal neuronal cell and is useful for the prevention or treatment of an ocular disease associated with retinal neuronal cell injury.

Hereinafter, examples of the preparation of the present invention and results of a pharmacological test will be described. However, these examples are described for the purpose of better understanding the present invention and are not intended to limit the scope of the present invention.

[Preparation Examples]

Hereinafter, examples of general preparations containing a prostaglandin F2a derivative according to the present invention will be described. 11 1) Ophthalmic Drop (in 100 mL)

Prostaglandin derivative F2a 10 mg Concentrated glycerin 2500 mg Polysorbate 80 2000 mg Monobasic sodium phosphate dihydrate 200 mg Sterile purified water q.s. 1 N hydrochloric acid or 1 N sodium hydroxide q.s. pH 6.0

An intended ophthalmic drop can be obtained by suitably altering the types and amounts of the prostaglandin F 2a derivative and additives. 2) Ophthalmic ointment (in 100g)

Prostaglandin derivative F2a 0.1 g Liquid paraffin 20 g Soft and white paraffin 77.9 g Purified lanolin 2 g

A desired ophthalmic ointment can be obtained by suitably altering the types and amounts of the prostaglandin F 2 O derivative and additives. 12 [Pharmacological Test]

In order to discover a new pharmaceutical application of a prostaglandin F2a derivative using rat fetal retinal neuronal cells, an effect of a prostaglandin F2cx derivative on the protection of retinal neuronal cells against retinal neuronal cell death induced by glutamate was evaluated and examined.

Incidentally, as the prostaglandin F2a derivative which is a test compound, 16-phenoxy-15-deoxy-15,15-difluoro-17,18,19,20-tetranor-prostaglandin F2a was used. (1) Culture of isolation of retinal neuronal cells

One pregnant SD rat underwent a laparotomy under systemic anesthesia and the uterus was transferred to a dish containing Hanks' balanced salt solution (HBSS). A fetal rat was isolated from the uterus and the ocular globes were removed from the rat fetus. The retina was isolated from the eyeballs under a stereoscopic microscope and cut into pieces with a scalpel. The retina was then further broken down to the cellular level and passed through a nylon mesh (No. 305, manufactured by NBC Industries Co., Ltd.) to remove cellular aggregates, and then the resulting filtrate was centrifuged at 1000 rpm for 4 minutes. The supernatant was removed and an appropriate amount of modified Eagle's medium (MEM) containing 10% fetal bovine serum (FBS) was added to the remaining cells to suspend them. After the cell count was counted with a hemocytometer, MEM medium containing 10% FBS was added thereto, whereby a cell suspension with a cell density of 0.8 x 106 cells / ml was obtained. The cell suspension was inoculated in an amount of 80 æL each into polyethyleneimine coated plastic discs and the discs were allowed to stand in an incubator (37øC, 5% CO 2). The day of cell inoculation was designated day 1 of culture and medium replacement was performed on all even number days. Incidentally, up to day 4, a MEM medium containing 10% FBS was used and after 8 days a MEM medium containing 10% horse serum (HS) was used. Incidentally, on day 6, a cytarabine-containing medium (Ara-C) (1.5 x 10-5 M in a MEM medium containing 10% FBS) was used for removal of proliferative cells. (2) Preparation of MEM medium containing HS containing test compound

2 mg of the test compound was dissolved in 100% ethanol and the resulting solution was sequentially diluted with a MEM medium containing HS, whereby a MEM medium containing HS containing the test compound was prepared at 0.1 nM, 1 nM, 10 nM or 100 nM. (3) Preparation of serum-free MEM medium containing test compound

2 mg of the test compound was dissolved in 100% ethanol and the resulting solution was sequentially diluted with a serum free MEM medium, whereby a serum free MEM medium containing the test compound was prepared at 0.1 nM, 1 nM, 10 nM or 100 nM. 14 (4) Evaluation of cell death

On day 10 of culture, the plastic disks in which the cells were inoculated and cultured were transferred to the HS containing MEM medium containing the test compound and incubated for 24 hours (37øC, 5% CO2). The plastic disks were transferred to a serum-free MEM medium containing 1 mM glutamate and incubated for 10 minutes and then transferred to the serum-free MEM medium containing the test compound and incubated for 1 hour (37øC, CO2 to 5%). The cells were then stained with a 1.5% trypan blue solution for 10 minutes and fixed by adding to this 10% formalin fixative solution. After the cells were washed with physiological saline, stained cells and uncolored cells were counted under an inverted microscope.

Incidentally, a vehicle delivery group was prepared by performing the same test as described above, except that a MEM medium containing HS was used in place of the above-mentioned MEM containing HS medium containing the test compound and a MEM medium free of serum medium instead of the above serum free MEM medium containing the test compound.

In addition, an untreated group was prepared by performing the same test as described above, except that a MEM medium containing HS was used in place of the above HS containing MEM medium containing the test compound and a serum free MEM medium in place of the above-mentioned serum-free MEM medium containing the test compound and, in addition, no treatment with a glutamate-containing serum free MEM medium was performed. The survival rate was calculated on the basis of the following calculation equation.

Survival rate (%) = {(number of cells not stained) / (number of cells not stained + number of cells stained)} x 100 (5) Results and discussion

As shown in Fig. 1, about 40% cell death of retinal neuronal cells was observed due to treatment with glutamate in the vehicle addition group. However, when the HS containing MEM medium containing the test compound (0.1 nM at 100 nM) was used as media, glutamate induced retinal neuronal cell death was inhibited in a concentration dependent effect, and it was confirmed that compound has a protective action of a retinal neuronal cell. Fig. 1 is a graph showing the survival rate for each concentration in the case of use of the test compound by the addition of glutamate.

Lisbon, August 30, 2012 16

Claims (1)

A compound represented by the following general formula (1) or a salt thereof for use in the prevention or treatment of an ocular disease associated with retinal neuronal cell injury, wherein the ocular disease is an abnormal field of vision, retinal vascular occlusion, diabetic retinopathy, ischemic optic neuropathy, macular degeneration, retinitis pigmentosa or Leber's disease: HQ (D wherein R represents a carboxyl group or a salt thereof or a (C1 -C6) alkoxycarbonyl group.